Corticotropin-releasing factor (CRF), urocortins (UCNs) and their receptors play a central role in the physiology of the hypothalamic pituitary adrenal axis, the central nervous system and a variety of peripheral tissues. What structural properties of the receptors are responsible for ligand selectivity and receptor activation? In Preliminary Results we described an oxidation-resistant radio labeled sauvagine analog (YQLS), that can be covalently cross linked to CRFR1 and CRFR2, using a chemical cross linking agent that cross links lysine residues. CNBr cleavage and site-directed mutagenesis were used to characterize lysine residues of the receptor that are in a close proximity to lysine residues of the bound ligand. Additionally, several p-benzoylphenylalanine (Bpa)-substituted YQLS radioligands, with a high affinity to CRFR1 and CRFR2, were developed and shown to cross-link to CRFR1 and CRFR2 after UV irradiation. Characterization of the important functional domains that are involved in ligand binding and signaling is an important question for understanding how ligand and receptor interact. We shall perform a fine mapping to characterize the residues (on the receptor) that are involved in the interaction with the ligand (Specific Aim 1). In Specific Aim 2 we shall perform a detailed map of the points of proximity between the bound ligand and its receptor using a combination of photo-affinity cross linking of Bpa-substituted radio labeled YQLS ligands and chemically-cross linked radio labeled YQLS. The CRF receptors have highly conserved 6 cysteine residues. We have analyzed the functional relevance of single and paired mutations of the cysteine residues and proposed a model for their disulfide bridge pairing. Recently, a new model was proposed from studying the chemical properties of a truncated NT- domain expressed in bacteria and in mammalian cells. In Specific Aim 3 we will examine the validity of the proposed model in mammalian cell lines expressing native receptors. Success in pursuing the objectives of this project will increase our understanding of the mechanisms by which CRF and urocortins bind to and activate their receptors and shall help the design and the development of receptor-specific agonists and antagonists.